Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation

doi:10.1016/S0002-9378(97)70302-8

American Journal of Obstetrics and Gynecology

Volume 177, Issue 4, October 1997, Pages 964-972

https://doi.org/10.1016/S0002-9378(97)70302-8 Get rights and content

Abstract

OBJECTIVE: Trophoblast differentiation is associated with externalization of phosphatidylserine from the inner to the outer surface of the plasma membrane. In this study we tested the hypothesis that concurrent externalization and binding of annexin-V blocks the phosphatidylserine-rich surface from acting as a site for activation of coagulation and that antiphospholipid antibodies lead to a procoagulant state by preventing annexin-V binding.

STUDY DESIGN: A choriocarcinoma model of trophoblast differentiation, forskolin-activated BeWo cells and immunoperoxidase techniques were used to determine surface and cytoplasmic localization of annexin-V related to differentiation. Monoclonal immunoglobulin M antibodies against phosphatidylserine- and cardiolipin-dependent antigens were used to determine the effects of antiphospholipid antibodies on annexin-V localization and on the binding of prothrombin to the BeWo surface.

RESULTS: During differentiation BeWo cells externalized phosphatidylserine and increased the expression of surface annexin-V. Monoclonal antibody against phosphatidylserine removed annexin-V from the BeWo surface and increased binding of prothrombin.

CONCLUSION: Antiphosphatidylserine antibody induces sites for prothrombin binding on the surface of a BeWo model of trophoblast, most likely by removing annexin-V. This mechanism could explain the frequent observation of increased thrombosis at the maternal-fetal interface in miscarriages associated with antiphospholipid antibodies. (Am J Obstet Gynecol 1997;177:964-72.)

Section snippets

BeWo choriocarcinoma cells

BeWo choriocarcinoma cells were maintained in Nutrient Mixture F-12 Ham (Sigma, St. Louis) with 15% fetal bovine serum (Sigma), 160 mg/dl glucose (Fisher, Pittsburgh, Pa.), 5 ml of 200 mmol/L l-glutamine (Sigma), and 5 ml of penicillin-streptomycin solution (Sigma). Cells were grown as monolayers in flasks at 37° C with 5% carbon dioxide. BeWo cells were split with use of trypsin–ethylenediaminetetraacetic acid solution, and 50 μl were plated onto 10-well glass microscope slides at a density of

Results

Forskolin induces differentiation of BeWo cells, which was confirmed by the production of hCG (Fig. 1, A).

. Verification of forskolin-induced differentiation of BeWo cells. A, Production of hCG. Forskolin-induced differentiation of BeWo cells resulted in production and secretion of the hormone hCG, detected on unfixed BeWo cells by immunoperoxidase labeling with anti-hCG. Reaction was graded as 2+. B, Externalization of phosphatidylserine-dependent antigens. Forskolin also induced

Comment

Phosphatidylserine in the plasma membrane is preferentially distributed to the inner leaflet through the action of a membrane aminophospholipid translocase (flippase).¹² Movement of large quantities of phosphatidylserine to the outer leaflet occurs relatively rarely but usually has significant repercussions. Erythrocytes undergoing senescence externalize phosphatidylserine, which is recognized by macrophage receptors, resulting in phagocytic removal of the aged red blood cell.¹³ Lymphocytes and

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Assessment of annexin A5 and annexin A2 levels as biomarkers for pre-eclampsia: A pilot study
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Deficient anticoagulant activity of annexin A5 and deficient profibrinolytic activity of annexin A2 have been linked to increased risk of thrombotic events. Placental dysfunction due to fibrin deposition/microthrombi has been implicated in the pathogenesis of pre-eclampsia (PE). In this study, we aimed to assess serum levels of annexin A5 and annexin A2 in a cohort of PE patients and investigate their role as biomarkers for the development of the disease. We examined 80 women in total; 40 healthy pregnant women and 40 pregnant women with PE after 20 weeks of pregnancy. Women were subjected to full clinical assessment, ultrasonography, and laboratory testing including complete blood picture, liver and kidney function tests and assessment of serum and urine proteins. Annexin A5 and annexin A2 were analyzed using enzyme-linked immunosorbent assay. The study showed serum annexin A2 but not annexin A5 was significantly reduced (P = 0.029) in women with PE (total and severe cases) compared to those with normal pregnancy. The ROC analysis of annexin A2 level for the prediction of development of PE showed an area under the curve of 0.64 (P = 0.029), and the best cut-off value was 0.89 ng/ml with a sensitivity of 70.0% and a specificity of 70.0%. Univariate analysis showed annexin A2 of <0.89 ng/ml, proteinuria, lower platelet count and higher BP were associated with significantly higher risk to develop PE. Based on this pilot study, serum annexin A2 levels may be a useful biomarker for pre-eclampsia. However, a larger study is required before a final conclusion is made.
Histopathology in the placentae of women with antiphospholipid antibodies: A systematic review of the literature
2015, Autoimmunity Reviews
Citation Excerpt :
However, perivillous/intervillous fibrin deposition and intervillous thrombi were not common features of the placentae of aPL-positive women, which questions the role of placental thrombosis in aPL-related obstetric morbidity. Proponents of the anticoagulant shield theory have demonstrated in vitro that aPLs displace phosphatidylserine-bound annexin V from the surface of the syncytiotrophoblast and decreases the time taken to form a clot in cell-surface coagulation [44–49]. However, the studies investigated in this systematic review have demonstrated that the expression of annexin V in the placentae of aPL-positive women can be less than [45,46], similar to [71,85], or greater than [84] the expression of annexin V in the placentae of aPL-negative control women.
Antiphospholipid antibodies (aPLs) are a heterogenous group of autoantibodies associated with recurrent miscarriage, stillbirth, foetal growth restriction and premature birth. The cause of obstetric morbidity in women with aPLs is not completely understood. Workers have attempted to understand the role of aPLs in obstetric morbidity by investigating the histopathology of placentae from aPL-positive women. However, it is unclear from these diverse, and at times contradictory reports what histopathological lesions are common in the placentae of women with aPLs. This systematic review was undertaken to generate a complete picture of the placental features associated with aPLs in an attempt to understand the pathological processes that occur in pregnancies affected by aPLs.
Pubmed, Scopus, Web of Science and Embase were searched on the 27th November 2014 using the keywords “placenta” OR “trophoblast” AND “antiphospholipid antibody” OR “antiphospholipid antibody syndrome”. Records that were relevant and eligible were qualitatively assessed and given a score out of 24.
Of the 1112 records that were retrieved from the systematic search, 34 records were eligible for review, and were qualitatively scored. Of the 44 histopathological features that were reported in 580 placentae from aPL-positive women, six features appeared to be more common in the placentae of aPL-positive women compared to control women, including: placental infarction, impaired spiral artery remodelling, decidual inflammation, increased syncytial knots, decreased vasculosyncytial membranes and the deposition of complement split product C4d.
Based on the evidence in this systematic review, a human placental aPL fingerprint has been proposed. The diversity of the human placental aPL fingerprint suggests that multiple pathological processes may occur in pregnancies affected by aPL.
Recurrent pregnancy loss: Evaluation and treatment
2015, Obstetrics and Gynecology Clinics of North America
Evaluation and treatment of recurrent pregnancy loss: A committee opinion
2012, Fertility and Sterility
Hydroxychloroquine reduces binding of antiphospholipid antibodies to syncytiotrophoblasts and restores annexin A5 expression
2011, American Journal of Obstetrics and Gynecology
Antibody-mediated disruption of the annexin A5 anticoagulant shield has been posited to be a thrombogenic mechanism in the antiphospholipid syndrome. We recently showed that the antimalarial drug, hydroxychloroquine, dissociates antiphospholipid immune complexes and restores annexin A5 binding to planar phospholipid bilayer. Using quantitative immunoassays, we demonstrated similar effects on BeWo trophoblasts. We therefore, investigated the effects of the drug on localization of annexin A5 in primary cultures of human placental syncytiotrophoblasts.
Laser confocal microscopy with computer-based morphometric analysis was used to localize annexin A5 and antiphospholipid antibodies on syncytiotrophoblasts exposed to polyclonal and monoclonal antiphospholipid and control immunoglobulin-Gs.
Hydroxychloroquine reversed the effects of the antiphospholipid antibodies on the syncytiotrophoblasts by markedly reducing immunoglobulin-G binding and restoring annexin A5 expression.
These results provide the first morphologic evidence for this effect of hydroxychloroquine on human placental syncytiotrophoblasts and support the possibility of novel treatments that target antiphospholipid antibody binding.
Resistance to annexin A5 anticoagulant activity in women with histories for obstetric antiphospholipid syndrome
2011, American Journal of Obstetrics and Gynecology
The objective of the study was to investigate whether resistance to annexin A5 anticoagulant activity (AnxA5) occurs in women with histories for obstetric complications of antiphospholipid syndrome (Obs-APS) and whether this correlates with antibody recognition of domain 1 of β2-glycoprotein.
One hundred thirty-six women with antiphospholipid antibodies, including 70 with histories for Obs-APS and 30 controls, were investigated.
Women with Obs-APS showed resistance to AnxA5 activity (median, 216%; range, 130–282% vs controls; median, 247%; range, 217–283%; P < .0001) and elevated levels of anti-domain I immunoglobulin (Ig) G (optical density: median, 0.056; range, 0.021–0.489 vs median, 0.042; range, 0.020–0.323; P = .002). Those in the lowest tertile of AnxA5 anticoagulant ratios had an odds ratio for Obs-APS of 58.0 (95% confidence interval, 3.3–1021.5). There was an inverse correlation between levels of annexin A5 anticoagulant activity and anti-domain I IgG.
Resistance to AnxA5 anticoagulant activity is associated with antibody recognition of domain I of β2-glycoprotein I and identifies a subset of women with histories for Obs-APS.

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^☆: From the Departments of Microbiology and Immunology^a and Obstetrics and Gynecology,^b Wright State University School of Medicine, and the Department of Applied Medical Sciences, University of Southern Maine.^c

^☆☆: Supported by National Institutes of Health grant No. HD23697 and a grant from the American Heart Association, Ohio Branch.

^★: Reprint requests: Neal S. Rote, PhD, Department of Microbiology and Immunology, School of Medicine, Wright State University, Dayton, OH 45435.

^★★: 0002-9378/97 $5.00 + 0 6/1/84229

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Antiphosphatidylserine antibody removes annexin-V and facilitates the binding of prothrombin at the surface of a choriocarcinoma model of trophoblast differentiation☆,☆☆,★,★★

Abstract

Section snippets

BeWo choriocarcinoma cells

Results

Comment

J Reprod Immunol

Am J Obstet Gynecol

Am J Obstet Gynecol

J Biol Chem

Thromb Res

Exp Cell Res

Clin Immunol Immunopathol

Biochim Biophys Acta

Placenta

J Biol Chem

J Reprod Immunol

Am J Obstet Gynecol